The nuclear enzyme topoisomerase (topo) II is required for cell division. Topo II is also the target of a variety of antineoplastic drugs which stabilize the topo II-DNA interaction at the site of enzyme-mediated strand passage leading to cell death. Drug-resistant forms of topo II have been isolated from drug-resistant cells. Genes coding these aberrant topo II's have been isolated and mutations within them putatively explain the cellular drug resistance. The work described in this proposal is aimed at proving or disproving the hypothesis that mutant enzyme forms completely explain the drug resistance exhibited by a human leukemia line induced to amsacrine resistance and a human brain tumor line that exhibits etoposide resistance despite having never been treated with etoposide. The gene coding for drug-sensitive topo II has been transfected into both resistant cell types. The drug-resistance of each has been partially overcome. To understand how this has occurred we will track the regulation of the expression of both this drug-sensitive gene (from Drosophila) and of the endogenous human topo II gene of each line. Drosophila and human topo II-specific gene probes and antibodies allow the independent tracking of exogenous vs. endogenous topo II gene product. This will allow us to see if the exogenous gene simply displaces or actually replaces the endogenous gene not only by expressing itself, but also by downregulating the endogenous topo II gene. If down- regulation of the endogenous gene occurs, the mechanism will be explored through the use of CAT constructs of the promoter region of the human topo II gene which will be transfected into the sensitized transfectants. The genes for any identified trans-acting regulatory factors will be cloned and sequenced. We have repeated the Drosophila topo II gene transfection using a human topo II gene. Drug-sensitization again accompanied exogenous topo II gene expression. Furthermore, a high-efficiency adenoviral vector containing the Drosophila topo II gene also gave this result. It must be remembered that even though this sensitization is only 2-3-fold, this is of sufficient magnitude to be clinically significant as it is within the range currently used to rationalize high dose therapy with autologous bone marrow or stem cell rescue. Principles elucidated in the work described above will be tested for their clinical relevance in the course of a clinical trial of the topo I-directed agent Topotecan in conjunction with other drugs including etoposide. This will allow us to test the utility of a newly devised filter binding assay that measures the drug-reactivity of the topo from circulating leukemic cells, and to test whether any of the regulatory factors governing topo II expression in the model cell systems are operating in patients. This should allow the development of assays having prognostic significance in the treatment of acute leukemia with topo- reactive agents and which could also serve as intermediate end-points in the performance of subsequent clinical trials.